Host and symbiont determinants of colonization by a co-evolved gut community Project Summary Host-specific gut bacteria are central to the biology and health of animals, but elucidating the processes that govern normal and atypical assembly of gut communities is challenging. Most gut communities, and specifically those of humans, are dauntingly complex; whereas others, such as that of Drosophila, have variable compositions dominated by opportunistic bacterial species from other environments. In the honey bee (Apis mellifera), the ileum region of the hindgut contains a dense but simple community dominated by only three specialized bacterial species that comprise >95% of bacteria despite the continual entry of diverse environmental microbes present in food. These three species are found only in bee guts, and distinct but related strains are found in related bee species. Our pilot work established axenic culture conditions, official nomenclature, and reference strains for all bee gut community members, transposon-based mutagenesis methods, protocols for controlling colonization with specific isolates or mutants, and methods for quantitative description of community composition. Furthermore, the host is amenable to genetic studies: sequenced genome and associated resources are available for the honey bee, and RNAi methods have been established. Building on these foundations, we will use the honey bee ileum as a model gut community to determine the specific bacterial and host factors that underlie the establishment of a persistent gut community characteristic of a host species. We will use expression analyses and genome-wide mutagenesis and mutant screening based on high-throughput sequencing to identify symbiont genes affecting colonization of the bee ileum and to elucidate the effects of host genes and pathways on colonization. We will examine how a focal pioneer symbiont influences the assembly and subsequent composition of the gut community. This will be achieved by monitoring succession in experimental gut communities and by identifying symbiont genes that affect colonization by other bacterial species and strains, including both the symbionts normally present in the community and the opportunistic or pathogenic bacteria that are typically excluded. These results will reveal host and symbiont-based processes that govern development of a normal, host- specific gut microbiota and will illuminate how and why gut communities sometimes develop abnormally, to the detriment of host health. !